The purpose of this research is to investigate the molecular mechanisms of action of biologically active proteins from arthropod disease vectors and pathogenic microorganisms. We use biological and physical techniques to characterize and understand the modes of action of pharmacologically active components from the saliva of blood-feeding vector insects and ticks, as well as immunomodulatory components secreted by parasitic organisms such as Toxoplasma and Schistosoma. [unreadable] [unreadable] Proteins and small molecules found in the saliva of vectors inhibit the host hemostatic responses and are essential for the successful completion of a blood meal. Most vector borne diseases are transmitted during feeding, so elucidation of the physiology and biochemistry of this process is necessary for understanding disease transmission. Saliva has also been shown to have pronounced effects on host inflammatory and immune responses which persist after feeding and can dramatically alter the environment for the pathogen after transmission. Determining the specific role of salivary molecules in these processes is essential for the understanding their importance to pathogen survival after transmission [unreadable] [unreadable] Over the past several years we have identified the functions of numerous salivary molecules involved primarily in overcoming host hemostatic defenses. The raw material for these studies comes from the analyses of salivary transcriptomes produced in collaboration with Dr. Jose Ribeiro. Bioinformatic analysis of sequence data is used to predict function of salivary proteins. Candidate proteins are then expressed in bacterial or eukaryotic cell systems. The proteins are purified and assayed using a variety of methods. Functionally characterized proteins are then produced in larger quantity for structural and other biophysical studies.[unreadable] [unreadable] Over this same period we have collaborated with Dr. Alan Sher's laboratory to characterize a number of pathogen-produced proteins involved in immune responses to infection. These projects included: The isolation of a T cell antigen from a Helicobacter species that is involved in the induction of colitis in a mouse model, the characterization of a chemokine receptor ligand from Toxoplasma which was evaluated for potential as an anti-retroviral agent, the isolation of a toll-like receptor ligand from Toxoplasma, and the isolation of an apparent T cell polarizing factor from the eggs of Schistosoma.[unreadable] [unreadable] During the past fiscal year we have 1) integrated protein structural analysis as a major component of our program, 2) characterized the mechanism of a potent salivary immunomodulatory protein from a tick species, 3) Determined the repertoire of salivary proteins from the rat flea by analyzing its salivary transcriptome, and 4) isolated a T cell polarizing factor from the eggs of the parasite Schistosoma mansoni. [unreadable] [unreadable] 1) We are now regularly crystallizing proteins in the laboratory and are making data-collection visits to the Advanced Photon Source synchrotron facility at Argonne Natl. Laboratory. We have produced recombinant protein, crystallized and determined the structures of two new proteins and have determined additional structures of these proteins to evaluate various ligand complexes. The "short form" D7 proteins found in saliva of the African malaria vector mosquito act to inhibit host infammatory responses and platelet aggregation by binding the biogenic amines serotonin, histamine and norepinephrine. We have determined the X-ray crystal structure of one of these proteins using multiple anomalous dispersion techniques as well as structures of the protein in complex with the ligands listed above. We have also determined the structure of the "long-form" D7 protein from the yellow fever mosquito which is a two-domain where one domain functions as a biogenic amine-binding protein while the function of the second domain is under investigation. The "long form" structure revealed a major confomational change that appears to be involved in the stabilization of bound ligands. As a potential practical application of this work, the short-form and long-form D7 proteins are being developed in collaboration with Dr. Loren Looger of HHMI Janelia Farms as biosensors for nerurotransmitters in neurological research. Along the same lines,we are working on the of two novel proteins serving as biogenic-amine binding proteins in thhe saliva of ticks. These proteins belong to the lipocalin protein familiy making them structurally unrelated to the mosquito D7 proteins. This information is being used to understand the process of blood feeding and the evolution of ticks.[unreadable] [unreadable] 2) Salivas of Ixodes (Lyme borreliosis vectors) ticks contain proteins aimed at supressing the immune system during the long feeding period of these species. We have examined the effects of two small proteins, sialostatin L and L2 which act to inhibit cathepsin type cysteine proteases. These molecules inhibit antigen presentation and inflammation. Supression of salivary expression of these proteins by RNAi results in profound growth inhibition of feeding ticks. We are examining the potential of immunization with this molecule to interfere with tick feeding and disease transmission. We are also pursuing structural studies with sialostatins L and L2 and their complexes with cathepsins.[unreadable] [unreadable] 3) The rat flea, Xenopsylla cheopis is a vector of the bubonic plague bacteium, Yersinia pestis. In order to study the biochemistry of blood feeding by this vector insect we have examined, in collaboration with Jose Ribeiro its entire repertoire of salivary proteins by sequencing and annotating its salivary transcriptome. Because this is the first member of its taxonomic order to be examined, a number of novel salivary molecules have been revealed, including a family related to scorpion toxins, and a family of phosphatase-like proteins which appear not to be active enzymes.[unreadable] [unreadable] 4) It was noted a number of years ago, that extracts of Schistosoma mansoni eggs induce polarization of CD4+ T cells toward a Th2 phenotype. The mechanism of Th2 polarization is not well understood and no specific factor inducing this differentiation has been isolated .In collaboration with Alan Sher and Dragana Jankovic of the Lab. of Parasitic Diseases, we have fractionated supernatants of egg cultures, and isolated an apparent single protein component which causes this effect. This protein has been expressed in human HEK 293 cells, and the recombinant material shows activity as indicated by increased populations of IL-4 positive CD4+ T cells in an in-vitro assay. We are currenly scaling up the production of the recombinant protein for further studies.